Fluroescence scanning images of cells labelled with rosamine derivatives
All eukaryotic cells, including plant cells and human cells, have a cell membrane, a cytoplasm and a nucleus. The cell membrane envelops the cell, separating the cytoplasm and the nucleus from the external environment; the cytoplasm holds organelles, such as mitochondria, ribosomes, the endoplasmic reticulum and the Golgi apparatus, in a translucent liquid called cytosol; and the nucleus stores DNA and RNA—the cell’s genetic blueprints.
Scientists involved in live-cell imaging often use small fluorescent molecules called fluorophores to ‘light up’ the cells. These fluorophores attach themselves to target proteins inside the cell and give off light when excited. Conventional fluorophores are non-specific, which means they bind to many different types of proteins inside the cell. The latest fluorophores, however, can be very specific and are capable of distinguishing proteins of one organelle from another.
Young Tae Chang at the A*STAR Singapore Bioimaging Consortium and co-workers have recently developed rosamine derivatives for detecting differentiated myotubes—tubular cells that eventually mature into muscle fibers. The rosamine derivatives give off light that is 2.3-fold brighter when they bind differentiation markers in myotubes (see image).
Chang and his co-workers wanted to identify the actual protein that the rosamine derivatives bind. At first, the researchers used an affinity pull-down assay—the conventional method for identifying small-molecule-binding proteins—to show that rosamine derivatives bind to tubulin, a protein found in the cytosol. Under the microscope, however, they observed that rosamine derivatives are concentrated in the mitochondria.
To resolve this mystery, the researchers made a chemical-affinity probe called CDy2 that is structurally similar to the rosamine derivatives. The advantage of using CDy2 is that it forms covalent bonds with its target proteins in the living cell. This means that even when target proteins are denatured, the researchers can still visualize them on gel under a fluorescence scanner.
The researchers disrupted the cell membrane of myotubes, collected the myotube’s internal content in a solution, and separated the proteins that bind CDy2. To their surprise, they found that CDy2 was bound to mitochondrial aldehyde dehydrogenase and not tubulin.
The researchers suggest that CDy2 is sequestered in the mitochondria before having a chance to react with tubulin in the cytosol. The finding shows that small molecules that bind one protein in vitro may or may not bind the same protein in vivo. It also reminds other researchers to be careful in their analysis of small-molecule-binding proteins as the results may differ in the complex environment of the living cell.
The A*STAR-affiliated researchers contributing to this research are from the Singapore Bioimaging Consortium.
